Chromatography

DEFINITION

CHROMATOGRAPHY

The separation of a mixture by distribution of its components between a mobile and stationary phase over time

– mobile phase = solvent

– stationary phase = column packing material

HISTORY

• Chromatography (from Greek :chromatos -- color ,

"graphein" -- to write) • 1903 Tswett - plant pigments separated

on chalk columns • 1931 Lederer & Kuhn - LC of

carotenoids • 1938 TLC and ion exchange • 1950 Reverse phase LC • 1954 Martin & Synge (Nobel Prize) • 1959 Gel permeation • 1965 instrumental LC (Waters)

Purpose of Chromatography

• Analytical - determine chemical composition of a sample

• Preparative - purify and collect one or more components of a sample

Uses for Chromatography

Real-life examples of uses for chromatography:

• Pharmaceutical Company – determine amount of each chemical found in new product

• Hospital – detect blood or alcohol levels in a patient’s blood stream

• Law Enforcement – to compare a sample found at a crime scene to samples from suspects

• Environmental Agency – determine the level of pollutants in the water supply

• Manufacturing Plant – to purify a chemical needed to make a product

Classification of Methods

There are two classification schemes:

– mobile phase

– attractive forces

Mobile Phase

• Gas (GC)

• Water (LC)

• Organic solvent (LC)

• Supercritical fluid (SCFC)

Classification based on

Mobile Phase

Gas Chromatography

Gas - solid Gas - liquid

Stationary Phase

Classification based on

Mobile Phase

Liquid chromatography (LC)

Column (gravity flow) High performance

(pressure flow)

Thin layer (adsorption)

Classification based on

Attractive Forces

1. Adsorption 2. Ion Exchange 3. Partition 4. Size Exclusion

Adsorption Chromatography

Separation based on their adsorption onto the surface of solid (stationary phase).

Normal phase-like separation

– Nonpolar mobile phase

for polar non-ionic compounds

Ex; Column chromatography

Partition Chromatography

solute are separated based on their partition between a liquid mobile phase and a liquid stationary phase coated on a solid support. – Normal – analyte is nonpolar organic;

stationary phase MORE polar than the mobile phase

– Reverse – analyte is polar organic; stationary phase LESS polar than the mobile phase

– Ex : TLC, Paper Chromatography

Phase 2

Phase 1

Phase 2

Phase 1

Ion Exchange Chromatography

Use ionic stationary phase – ions separated on the basis of their tendency to

displace counter ions adsorbed on stationary phase (Depends on charge, hydration, “solubility”…)

Anionic stationary phases: used for cation separation Cationic stationary phases : for anion separation for ionic compounds - Ex : CC (K.turus), HPLC

Size Exclusion Chromatography

Separation is a result of “trapping” of molecules in the pores of the packing material

• Very large molecules can’t get

into the pores – unretained • Very small molecules get hung up

in to pores for a long time - most retained – longest retention time

• stationary phase is a porous matrix

• Ex: CC, HPLC

• Liquid Chromatography – separates liquid samples with a liquid solvent (mobile phase) and a column composed of solid beads (stationary phase)

• Gas Chromatography – separates vaporized samples with a carrier gas (mobile phase) and a column composed of a liquid or of solid beads (stationary phase)

• Paper Chromatography – separates dried liquid samples with a liquid solvent (mobile phase) and a paper strip (stationary phase)

• Thin-Layer Chromatography – separates dried liquid samples with a liquid solvent (mobile phase) and a glass plate covered with a thin layer of alumina or silica gel

(stationary phase)

Types of Chromatography

STATIONARY PHASE

Type of chromatography Material

Paper chromatography

(KK = kertas kromatografi)

Filter paper, cellulose

Thin Layer Chromatography

(KLN = Kromatografi lapisan nipis)

Silica gel, alumina, polyamide

Gas chromatography

(GC)

Squalene, apezion, carbowax M

High Performance Liquid Chromatography

(KCPT = kromatografi cecair prestasi tinggi)

C-8, C-18, Licosorb, Silicone

MOBILE PHASE

Type of chromatography Solvent

Paper chromatography

(KK = kertas kromatografi)

Air, alcohol

Thin Layer Chromatography

(KLN = Kromatografi lapisan nipis)

Hexane, ether petroleum, alcohol.

Gas chromatography

(GC)

He, Ar, N2

High Performance Liquid Chromatography

(KCPT = kromatografi cecair prestasi tinggi)

Cyclohexane, n-hexane, carbon tetrachloride, ethanol, methanol, air